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Background & Objectives American foulbrood (AFB), caused by the highly virulent, spore-forming bacterium Paenibacillus larvae, poses a significant threat to honey bee brood. The widespread use of antibiotics not only fails to effectively combat the disease but also raises concerns regarding honey safety. The current computational study was attempted to identify a novel therapeutic drug target against P. larvae, a causative agent of American foulbrood disease in honey bee. Methods We investigated effective novel drug targets through a comprehensive in silico pan-proteome and hierarchal subtractive sequence analysis. In total, 14 strains of P. larvae genomes were used to identify core genes. Subsequently, the core proteome was systematically narrowed down to a single protein predicted as the potential drug target. Alphafold software was then employed to predict the 3D structure of the potential drug target. Structural docking was carried out between a library of phytochemicals derived from traditional Chinese flora (n > 36,000) and the potential receptor using Autodock tool 1.5.6. Finally, molecular dynamics (MD) simulation study was conducted using GROMACS to assess the stability of the best-docked ligand. Results Proteome mining led to the identification of Ketoacyl-ACP synthase III as a highly promising therapeutic target, making it a prime candidate for inhibitor screening. The subsequent virtual screening and MD simulation analyses further affirmed the selection of ZINC95910054 as a potent inhibitor, with the lowest binding energy. This finding presents significant promise in the battle against P. larvae. Conclusions Computer aided drug design provides a novel approach for managing American foulbrood in honey bee populations, potentially mitigating its detrimental effects on both bee colonies and the honey industry.

Marek’s disease virus (MDV) is a highly contagious and persistent virus that causes T-lymphoma in chickens, posing a significant threat to the poultry industry despite the availability of vaccines. The emergence of new virulent strains has further intensified the challenge of designing effective antiviral drugs for MDV. In this study, our main objective was to identify novel antiviral phytochemicals through in silico analysis. We employed Alphafold to construct a three-dimensional (3D) structure of the MDV DNA polymerase, a crucial enzyme involved in viral replication. To ensure the accuracy of the structural model, we validated it using tools available at the SAVES server. Subsequently, a diverse dataset containing thousands of compounds, primarily derived from plant sources, was subjected to molecular docking with the MDV DNA polymerase model, utilizing AutoDock software V 4.2. Through comprehensive analysis of the docking results, we identified Disalicyloyl curcumin as a promising drug candidate that exhibited remarkable binding affinity, with a minimum energy of −12.66 Kcal/mol, specifically targeting the DNA polymerase enzyme. To further assess its potential, we performed molecular dynamics simulations, which confirmed the stability of Disalicyloyl curcumin within the MDV system. Experimental validation of its inhibitory activity in vitro can provide substantial support for its effectiveness. The outcomes of our study hold significant implications for the poultry industry, as the discovery of efficient antiviral phytochemicals against MDV could substantially mitigate the economic losses associated with this devastating disease.

Soft rot disease in potato is a major problem in fields and warehouses all over the world. Although it is known that bacteria from the genera Pectobacterium and Dickeya are the main causative agents of soft rot diseases, recent studies indicate the involvement of pectinolytic Bacillus and Paenibacillus in this disease. In the present research, samples of potato with soft rot symptoms were collected from eight governorates of Tunisia. Two hundred and seventy bacterial isolates were acquired from tubers. Twenty of the isolated strains indicted pectinolytic activity by forming deep cavities on crystal violet pectate medium. All pectinolytic isolates were able to macerate potato tuber tissue. Phenotypic characterisation showed that these isolates were Gram-positive bacilli, exhibiting pectinolytic, cellulolytic, proteolytic and amylolytic activity. The majority of the isolates indicated swimming and swarming motility. The application of API test, MALDI-TOF MS and 16S rDNA sequencing allowed for the assignment of nineteen of the tested isolates to the species Bacillus pumilus and one to the species Paenibacillus amyloliticus. To the best of our knowledge, this is the first report of soft rot in potato caused by pectinolytic B. pumilus in Tunisia.

There is currently an increasing demand for the characterization of endophytic bacteria isolated from different parts of plants (rhizosphere, roots, fruit, leaf) in order to improve the organic agriculture practices. The current research was performed to identify both rhizospheric bacteria isolated from the rhizosphere of Ficus carica in three different sites in the north of Tunisia and endophytic bacteria isolated from dried figs. We then characterized them for a diversity of plant growth-promoting (PGP) activities. A collection of 120 isolates from rhizospheric soil and 9 isolates from dried figs was obtained and purified. 16SrDNA gene amplification of rhizospheric bacteria revealed significant diversity and allowed for the assigning of the isolates to 6 phyla: Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Representative strains of the collection (90 strains) were tested for numerous PGP activities and resistance to abiotic stresses. The most common PGP trait for all bacteria from the three regions was siderophore production (62%), followed by cellulase (38%), then protease activity (37%), then by lipases activity (17%) and lastly by solubilization of phosphates (9%). Twenty -three strains that showed most PGP traits were selected, 8 strains presented 12 or more, and 15 strains displayed between 7 and 11 of 17 PGP activities. The majority of the isolates manifested a possible adaptation to abiotic stress and unfavorable environments. PCR-DGGE analysis of soil rhizosphere of the three sites allowed also for the acquisition of a Cluster analysis of rhizospheric bacterial communities. Our current study identified and characterized for the first time in Tunisia rhizospheric and endophytic bacteria from dried fruit of Ficus carica.

Abstract The damages observed in Tunisian citrus orchards have prompted studies on the Pseudomonas spp. responsible for blast and black pit. Prospective orchards between 2015 and 2017 showed that the diseases rapidly spread geographically and to new cultivars. A screening of Pseudomonas spp. isolated from symptomatic trees revealed their wide diversity according to phylogenetic analysis of their housekeeping rpoD and cts genes. The majority of strains were affiliated to Pseudomonas syringae pv. syringae (Phylogroup PG02b), previously described in Tunisia. However, they exhibited various BOX-PCR fingerprints and were not clonal. This work demonstrated, for the first time in Tunisia, the involvement of Pseudomonas cerasi (PG02a) and Pseudomonas congelans (PG02c). The latter did not show significant pathogenicity on citrus, but was pathogenic on cantaloupe and active for ice nucleation that could play a role in the disease. A comparative phylogenetic study of citrus pathogens from Iran, Montenegro and Tunisia revealed that P. syringae (PG02b) strains are closely related but again not clonal. Interestingly P. cerasi (PG02a) was isolated in two countries and seems to outspread. However, its role in the diseases is not fully understood and it should be monitored in future studies. The diversity of pathogenic Pseudomonas spp. and the extension of the diseases highlight that they have become complex and synergistic. It opens questions about which factors favor diseases and how to fight against them efficiently and with sustainable means.

Pseudomonas syringae pv. syringae (Pss), the causal agent of citrus blast and black pit lesion of lemon fruit, continues to cause serious damage in citrus production in Tunisia. Faced with the rapid emergence of the disease and the inefficiency of conventional control methods, an alternative strategy based on the use of bacteriophages was pursued in this study. The lytic Pss bacteriophage SoKa was isolated from soil collected from Tunisian citrus orchards. Analysis of the host range showed that SoKa was able to lyse seven other Pss strains. Interestingly, Pseudomonas syringae pv. porri, pathogenic to leek, could also be infected by SoKa. The activity of SoKa was maintained at pH values between 2 and 10, at temperatures between −80 and 37 °C; the phage could resist UV radiation at an intensity of 320 nm up to 40 min. Whole genome sequencing revealed that the Pseudomonas phage SoKa is a novel phage that belongs to the Bifseptvirus genus of the Autographiviridae family. The absence of virulence proteins and lysogeny-associated proteins encoded on the phage genome, its anti-biofilm activity, and the significant reduction of tissue necrosis in different fruit bioassays make SoKa potentially suitable for use in phage biocontrol.

Overall, 180 yeasts and bacteria isolated from the peel of citrus fruits were screened for their in vitro antagonistic activity against Penicillium digitatum and P. italicum, causative agents of green and blue mold of citrus fruits, respectively. Two yeast and three bacterial isolates were selected for their inhibitory activity on mycelium growth. Based on the phylogenetic analysis of 16S rDNA and ITS rDNA sequences, the yeast isolates were identified as Candida oleophila and Debaryomyces hansenii while the bacterial isolates were identified as Bacillus amyloliquefaciens, B. pumilus and B. subtilis. All five selected isolates significantly reduced the incidence of decay incited by P. digitatum and P. italicum on ‘Valencia’ orange and ‘Eureka’ lemon fruits. Moreover, they were effective in preventing natural infections of green and blue mold of fruits stored at 4 °C. Treatments with antagonistic yeasts and bacteria did not negatively affect the quality and shelf life of fruits. The antagonistic efficacy of the five isolates depended on multiple modes of action, including the ability to form biofilms and produce antifungal lipopeptides, lytic enzymes and volatile compounds. The selected isolates are promising as biocontrol agents of postharvest green and blue molds of citrus fruits.

Botrytis cinerea, the causal agent of grey mould, is a predominant agent causing extensive postharvest and quality losses of apples in Tunisia and worldwide. Efforts to manage this disease have met with limited success. For this reason, the use of microorganism preparations to control fungal diseases as an alternative to fungicides became an urgent need. From a total of 60 epiphytic yeasts, 10 were assessed in vitro against B. cinerea and selected isolates showing antagonism were evaluated for their ability to suppress the grey mould in vivo. On Petri plates, the most promising strains (three strains of Aureobasidium pullulans, one Cryptococcus flavescens, and one Citeromyces matritensis) showed a zone of inhibition against the pathogen fungus not exceeding 10 mm. In vivo, these isolates showed a remarkable antifungal activity since they significantly reduced disease severity on apples from 63% to 95% compared to the control. In conclusion, the work has demonstrated that the three strains, L7 of Aureobasidium pullulans, L2 of Citeromyces matritensis, and L10 of Cryptococcus flavescens, were highly effective and can be used as potential biocontrol agents in controlling the post-harvest decay of apples caused by B. cinerea.

Significant losses of clementine trees (Citrus × clementina) due to Phytophthora branch canker were observed in the Cap Bon Peninsula in northeastern Tunisia. This disease is caused by Phytophthora citrophthora. The low efficiency of available cultural and host resistance disease management methods, and potential harmful impacts of pesticide strategies, lead to a search of new control alternatives. This study investigated potential biocontrol agents. The halophilic bacterial strains Bacillus pumilus M3-16 and Halomonas elongata L80, previously selected for their antifungal activity, were assessed in a greenhouse trial. In addition, 69 endophytic bacteria were isolated from citrus roots and assessed for their antagonistic activities. Five isolates were selected because they showedstrong growth inhibition of P. citrophthora in dual culture tests. Based on phenotypic characteristics, biochemical tests and sequence analyses of the 16S region of rDNA, the bacteria were identified as Bacillus pumilus (S19), Bacillus amyloliquefaciens (S24), Bacillus siamensis (S54) Paenibacillus polymyxa (S31), and Pseudomonas veronii (S40). These metabolized different carbon sources, and possessed antibiotic genes, produced siderophores and phytohormones, and solubilized phosphates. In greenhouse trials, two endophytic strains (S24, S31) and two extremophile strains (M3-16, L80) were also assessed, these bacteria significantly reduced (P< 0.0001) necrotic lesions on host plants, by 35% for strain S24 of Bacillus amyloliquefaciens to 51% for strain L80 of Halomonas elongata. This is the first report of Halomonas sp. Controlling a Phytophthora sp. The strains described in this study offer a foundation for developing an efficient biofungicide.

Soft rot disease in potato is a major problem in fields and warehouses all over the world. Although it is known that bacteria from the genera Pectobacterium and Dickeya are the main causative agents of soft rot diseases, recent studies indicate the involvement of pectinolytic Bacillus and Paenibacillus in this disease. In the present research, samples of potato with soft rot symptoms were collected from eight governorates of Tunisia. Two hundred and seventy bacterial isolates were acquired from tubers. Twenty of the isolated strains indicted pectinolytic activity by forming deep cavities on crystal violet pectate medium. All pectinolytic isolates were able to macerate potato tuber tissue. Phenotypic characterisation showed that these isolates were Gram-positive bacilli, exhibiting pectinolytic, cellulolytic, proteolytic and amylolytic activity. The majority of the isolates indicated swimming and swarming motility. The application of API test, MALDI-TOF MS and 16S rDNA sequencing allowed for the assignment of nineteen of the tested isolates to the species Bacillus pumilus and one to the species Paenibacillus amyloliticus. To the best of our knowledge, this is the first report of soft rot in potato caused by pectinolytic B. pumilus in Tunisia. [ABSTRACT FROM AUTHOR]

Background. Foot mycoses are a frequent disease that represents a public health problem worldwide. Objectives. This study aims to evaluate the epidemiology of foot mycoses among Tunisian patients, in order to determine the fungal etiological agents and to identify possible risk factors. Patients and Methods. A prospective study of three hundred and ninety-two patients was undertaken during one year (2013-2014). All subjects were asked to collect demographic data related to the risk factors of foot mycoses. A complete mycological diagnosis was carried out on all patients. Results. A total of 485 samples were collected; tinea pedis and tinea unguium were confirmed in 88.2% of cases. Dermatophytes were isolated in 70.5% and the most frequent pathogen was Trichophyton rubrum (98.1%), followed by yeasts (17.7%) commonly Candida parapsilosis. Non-dermatophyte molds (NDMs) were observed in 8.02% cases and Fusarium sp. was the frequent genus (29.1%). The main predisposing factors of fungal foot infections were practicing ritual washing (56.6%) and frequentation of communal showers (50.5%). Conclusion. This is a recent survey of foot mycoses in Tunisia. Epidemiological studies can be useful to eradicate these infections and to provide further measures of hygiene and education.

Background: Bacterial resistance is currently a significant global public health problem. Acinetobacter baumannii has been ranked in the list of the World Health Organization as the most critical and priority pathogen for which new antibiotics are urgently needed. In this context, computational methods play a central role in the modern drug discovery process. The purpose of the current study was to identify new potential therapeutic molecules to neutralize MDR A. baumannii bacteria. Methods: A total of 3686 proteins retrieved from the A. baumannii proteome were subjected to subtractive proteomic analysis to narrow down the spectrum of drug targets. The SWISS‐MODEL server was used to perform a 3D homology model of the selected target protein. The SAVES server was used to evaluate the overall quality of the model. A dataset of 74500 analogues retrieved from the PubChem database was docked with LpxC using the AutoDock software. Results: In this study, we predicted a putative new inhibitor for the Lpxc enzyme of A. baumannii. The LpxC enzyme was selected as the most appropriate drug target for A. baumannii. According to the virtual screening results, N‐[(2S)‐3‐amino‐1‐(hydroxyamino)‐1‐oxopropan‐2‐yl]‐4‐(4‐bromophenyl) benzamide (CS250) could be a promising drug candidate targeting the LpxC enzyme. This molecule shows polar interactions with six amino acids and non‐polar interactions with eight other residues. In vitro experimental validation was performed through the inhibition assay. Conclusion: To the best of our knowledge, this is the first study that suggests CS250 as a promising inhibitory molecule that can be exploited to target this gram‐negative pathogen. [ABSTRACT FROM AUTHOR]